Involvement of ER stress in retinal cell death.

PURPOSE
To clarify whether endoplasmic reticulum (ER) stress is involved in retinal cell death, using cultured retinal ganglion cells (RGC-5, a rat ganglion cell line transformed with E1A virus), and transgenic mice ER stress-activated indicator (ERAI) mice carrying a human XBP1 and venus a variant of green fluorescent protein (GFP) fusion gene.


METHODS
RGC-5 damage was induced by tunicamycin, and cell viability was measured by double nuclear staining (Hoechst 33342 and either YO-PRO-1 or propidium iodide). The expressions of glucose-regulated protein 78(GRP78)/BiP, the phosphorylated form of eukaryotic initiation factor 2alpha (p-eIF2alpha), and C/EBP-homologous (CHOP) protein after tunicamycin (in vitro or in vivo) or N-methyl-D-aspartate (NMDA; in vivo) treatment were measured using immunoblot or immunostaining. ERAI mice carrying the F-XBP1-DBD-venus expression gene were used to monitor ER-stress in vivo. Twenty-four hours after intravitreal injection of tunicamycin or NMDA, or after raising intraocular pressure (IOP), the retinal fluorescence intensity was visualized in anesthetized animals using an ophthalmoscope and in retinal flatmount or cross-section specimens using laser confocal microscopy.


RESULTS
Treatment with tunicamycin induced apoptotic cell death in RGC-5 and also induced production of ER stress-related proteins (BiP, the phosphorylated form of eIF2alpha, and CHOP protein). In vivo, tunicamycin induced retinal ganglion cell (RGC) loss and thinning of the inner plexiform layer, 7 days after intravitreal injection. In flatmounted retinas of ERAI mice, the fluorescence intensity arising from the XBP-1-venus fusion protein, indicating ER-stress activation, was increased at 24 h after tunicamycin, NMDA, or IOP elevation. In transverse cross-sections from ERAI mice, the fluorescence intensity was first increased in cells of the ganglion cell and inner plexiform layers at 12 and 24 h, respectively, after NMDA injection, and it was localized to ganglion and amacrine cells at 12 and 24 h, respectively, and to microglial cells at 72 h. BiP and CHOP were increased at 12 h after NMDA injection, and the increases persisted for the remainder of the 72 h observation period.


CONCLUSIONS
These data indicate that ER-stress may play a pivotal role in RGC death, whether induced by NMDA or IOP elevation.

delivered via an animal general anesthesia machine (Soft Lander, Sin-ei industry Co. Ltd., Saitama, Japan). The body temperature was maintained at 37.0 -37.5 °C with the aid of a heating pad and heating lamp. Retinal damage was induced by injection (2 µl/eye) either of NMDA (Sigma-Aldrich) at 20 mM dissolved in 0.01 M phosphate-buffered saline (PBS) or of tunicamycin at 50 and 500 µg/ml, or (b) by acutely increasing the intraocular pressure (IOP). For NMDA-or tunicamycin-induced injury, the relevant agent was injected into the vitreous body of the left eye under the above anesthesia. In the IOP elevation model, the pupils were dilated with topical 2.5% phenylephrine hydrochloride and 1% tropicamide (Santen Pharmaceuticals Co. Ltd., Osaka, Japan). After topical instillation of 0.4% oxybuprocaine hydrochloride (Santen Pharmaceuticals Co. Ltd.), the anterior chamber was cannulated with a 32-gauge needle connected to a reservoir containing 0.9% NaCl. IOP was elevated by raising the height of the reservoir, maintaining a pressure of 100 mm Hg for 45 min. Retinal ischemia was confirmed by the blanching of the iris and retinal circulation. At the end of the elevated IOP period, the needle was removed, and reperfusion of the retinal vasculature was confirmed by ophthalmoscopic examination (KOM 300; Konan Inc., Nishinomiya, Japan). One drop of levofloxacin ophthalmic solution (Santen Pharmaceuticals Co. Ltd.) was applied topically to the treated eye after each procedure (intravitreal injection or ischemia-reperfusion).
Monitoring endoplasmic reticulum (ER) stress using ERAI-transgenic mice: In anesthetized ERAI-transgenic or wild-type mice, retinal damage was induced by injection (2 µl/eye) of either NMDA at 20 mM or tunicamycin at 50 µg/ ml into the vitreous body, or by elevating IOP to 100 mmHg for 45 min (see above). Twenty-four hours later, the fluorescence intensity arising from the XBP-1-venus fusion protein, which is translated from the F-XBP1DDBD-venus gene, was visualized in the retina of anesthetized animals using an ophthalmoscope (TRC-50; TOPCON, Tokyo, Japan) fitted with a fluorescence filter. In separate experiments, the distribution and time-course of changes in fluorescence intensity in the retina were measured in retinal flatmount and cross-section specimens using either laser confocal microscopy (Bio-Lad Laboratories, Inc, Hercules, CA) or epifluorescence microscopy (Power BX50; Olympus, Tokyo, Japan). At various times after the intravitreal injections (12,24, and 72 h), eyes were enucleated, then fixed in 4% paraformaldehyde for 1 h or overnight at 4 °C as preparation for retinal flatmount and retinal cross-section, respectively. For the preparation of retinal flatmounts, detached retinas were flatmounted on slides (MAS COAT; MATSUNAMI GLASS IND., LTD., Osaka, Japan) by making radial incisions. They were then mounted under a coverslip and observed using the epifluorescence microscope. For the preparation of retinal cross-sections, fixed eyes were immersed in 20% sucrose for 48 h at 4 °C, and embedded in optimum cutting temperature (OCT) compound (Sakura Finetechnical Co., Ltd, Tokyo, Japan). Transverse, 10 µm thick cryostat sections were cut and placed onto slides (MAS COAT) under a coverslip, and observed using the laser confocal microscope.
Histological analysis of mouse retina: Seven days after the NMDA or tunicamycin injection, eyeballs were enucleated for histological analysis. In mice under anesthesia, produced by an intraperitoneal injection of sodium pentobarbital (80 mg/kg), each eye was enucleated, then kept immersed for at least 24 h at 4 °C in a fixative solution containing 4% paraformaldehyde. Six paraffin-embedded sections (thickness, 3 µm) cut through the optic disc of each eye were prepared in a standard manner, and stained with hematoxylin and eosin. Retinal damage was evaluated as described previously, and three sections from each eye were used for the morphometric analysis. Light-microscope photographs were taken using a digital camera (Coolpix 4500, Nikon) and the cell counts in the ganglion cell layer (GCL) and the thickness of the inner plexiform layer (IPL) at a distance between 350 and 650 µm from the optic disc were measured on the images in a masked fashion by a single observer (Y.I.). Data from three sections (selected randomly from the six sections) were averaged for each eye, and the values obtained were used to evaluate the GCL cell count and the IPL thickness.
Statistical analysis: Data are presented as the means±SEM. Statistical comparisons were made using a Student's t-test or Dunnett's test, by means of STAT VIEW version 5.0 (SAS Institute Inc., Cary, NC). P<0.05 was considered to be statistically significance.

Retinal cell death and time-course of changes in endoplasmic reticulum (ER) stress-related protein induced by tunicamycin:
We examined whether tunicamycin treatment could induce cell death through ER stress in retinal ganglion cell using RGC-5. Representative fluorescence stainings of nuclei [using Hoechst 33342, YO-PRO-1, and propidium iodide (PI) dyes] are shown in Figure 1A. Vehicle-treated control cells displayed normal nuclear morphology and negative staining with both YO-PRO-1 dye (which stains early apoptotic and later-stage cells) and PI dye (which stains late-stage apoptotic cells; upper panels in Figure 1A). Treatment with tunicamycin led to shrinkage and condensation of nuclei, and to positive staining with each of these dyes (lower panels in Figure 1A). The number of cells exhibiting PI fluorescence was counted, and positive cells were expressed as the percentage of PI-to Hoechst 33342-positive cells ( Figure 1B). After treatment with tunicamycin at 1, 2, or 4 µg/ml for 24 h, the percentages of PI-positive cells were 8.3±1.2% (n=6), 13.1±0.9% (n=6), and 11.3±0.6% (n=6), respectively, while in the non-treated control group the percentage was 0.5±0.2% (n=6). After treatment with tunicamycin at 1, 2, or 4 µg/ml for a longer time period (48 h), the corresponding values were 41.5±3.5% (n=6), 43.7±2.1% (n=6), and 50.7±2.6% (n=6), respectively (1.2±0.4% (n=6) for the nontreated control group). Time-course data for the changes in the protein levels of glucose-regulated protein (GRP)78/BiP, the phosphorylated form of eukaryotic initiation factor 2α (eIF2α), total eIF2α, and C/EBP-homologous protein (CHOP) occurring after tunicamycin treatment at 2 µg/ml are shown in Figure 1C. BiP, a biomarker of ER-stress, increased timedependently throughout the 24 h tunicamycin treatment period, while actin levels remained unchanged. Treatment with tunicamycin time-dependently induced eIF2α phosphorylation, while total eIF2α levels were not changed during the 24 h observation period. CHOP was first detected at 6 h after addition of tunicamycin and persisted thereafter. These data indicate that treatment with tunicamycin can induce expres-sions of ER stress-related proteins and subsequent apoptotic cell death in RGC-5 culture in vitro.
Intravitreal injection of tunicamycin induces retinal cell death in mice: To clarify whether tunicamycin would induce retinal cell death in vivo, we examined the histological changes in the retina at 7 days after intravitreal injection of tunicamycin. As shown in Figure 2, intravitreal injection of tunicamycin at 0.1 µg/eye (a low dose) induced a significant loss of cells in the retinal ganglion cell layer (GCL), but no thinning of the inner plexiform layer (IPL; versus vehicle-treated retinas). At a high dose of 1µg/eye, tunicamycin significantly decreased both the cell count in GCL and the IPL thickness (versus the non-treated normal retina; Figure 2). On the other hand, no retinal damage was induced by intravitreal injection of an identical volume of vehicle (versus the non-treated retina). Together, these findings suggest that tunicamycin at 0.1 µg/eye (giving an estimated concentration in the vitreous body of approximately 10 µg/ml) induces retinal ganglion cell death at a concentration similar to that inducing exhibiting the apoptotic cell death in RGC-5 in vitro.

Increase in XBP-1-venus fusion protein in the retina in ER stress-activated indicator (ERAI)-transgenic mice:
To investigate whether ER stress is induced in the mouse retina during the early stages of retinal damage in vivo, we used ERAI-transgenic mice carrying the F-XBP1DDBD-venus expression gene, which allows effective identification of cells under ER stress in vivo, as previously described by Iwawaki et al. [11]. Twenty-four h after intravitreal injection of tunicamycin at 0.1 µg or of N-methyl-D-aspartate (NMDA) at 40 nmol, the fluorescence intensity arising from the XBP-1-venus fusion protein was visualized in the retina of anesthetized animals (using an ophthalmoscope) as shown in    In the NMDA-treated retinas of ERAI mice, the background fluorescence intensity was time-dependently increased in the period from 12 to 72 h, but little change was observed in the NMDA-treated retinas of wild-type mice.
These changes in background could reflect increases in the lower part of the ganglion cell layer, such as the inner plexiform layer and neuroepithelial layer, of the retina. In transverse sections, increases in fluorescence intensity were first observed in cells of the GCL and inner plexiform layer at 12 and 24 h, respectively, after NMDA injection, and the increases peaked in GCL cells at 24 h ( Figure 4B). The increase in fluorescence had diminished at 72 h after the NMDA injection, but morphologically distinct cells (such as microglia cells) had appeared in GCL. On the other hand, the retinas of wild-type and non-treated ERAI mice showed a low fluorescence intensity (below background), while a slight fluorescence intensity was observed in the neuroepithelial layer of the retina ( Figure  4B). These cells merged with Thy-1-positive cells (ganglion cells) and some OX-42-positive cells (microglia) in GCL, and with HPC-1-positive cells (amacrine cells) in IPL ( Figure 4C). Together, these results suggest that XBP-1 splicing, representing activation of the ER-stress signal pathway, may be induced in retinal ganglion and amacrine and microglia cells during the early stages of retinal cell damage.
Increases in GRP78/BiP and CHOP in mouse retina after NMDA injection: To clarify whether ER stress-related proteins other than XBP-1 are induced in the mouse retina by NMDA stimulation, we examined the changes in BiP, a biomarker of ER stress, in the retina after intravitreal injection of NMDA. As shown in Figure 5B, cell loss in GCL and thinning of IPL were observed at 72 h after NMDA injection (versus non-treated control retinas; Figure 5A). Using immunoblots, as shown in Figure 5C, we found that BiP was significantly increased at 12 h after the NMDA injection, and that the increase persisted for the remainder of the 72 h observation period. Next, we investigated the distribution and timecourse of changes in GRP78/BiP and CHOP, a proapoptosis protein, after NMDA injection. In the non-treated control retina, slight immunoreactivities for BiP and CHOP were observed in a number of cells in GCL and IPL ( Figure 5D). Increases in these immunoreactivities were observed in retinal ganglion cells at 12 h after NMDA injection, and time-dependent increases were noted in the inner retina ( Figure 5D).

DISCUSSION
In the present study, we could detect pathological changes and time-dependent changes related to ER stress in retinal flatmount and transverse sections and in the retinas of living mice after retinal damage. Moreover, we demonstrated that ER stress signals were activated in the retina in vivo after tunicamycin, elevating IOP, or NMDA treatment.
Agents or conditions that adversely affect ER protein folding lead to an accumulation of unfolded or misfolded proteins in the ER, a condition defined as ER stress. ER stress can be induced by agents or conditions that interfere with (a) protein glycosylation (e.g., glucose starvation, tunicamycin, glucosamine), (b) disulfide-bond formation (e.g., DTT, homocysteine), (c) Ca 2+ balance (A23187, thapsigargin, EGTA), and/ or (d) a general overloading of the ER with proteins (e.g., viral or non-viral oncogenesis) [1,13,14]. However, little is known about any involvement of ER stress in retinal damage. In the present study, we found that tunicamycin induced the ER stress-associated proteins BiP, p-eIF2α, and CHOP in cultured RGC-5 cells. These protein levels started to increase at 2 to 6 h after the start of tunicamycin treatment, and increased time-dependently until 24 h after the start of the treatment, while apoptotic cell death with condensation and fragmentation of nuclei was observed 24 h later. BiP acts as an ER resident molecular chaperon that is induced by ER stress, and this protein refolds the unfolded proteins, thereby tending to maintain homeostasis in the ER [15,16]. Since CHOP is a member of the CCAAT/enhancer-binding protein family that is induced by ER stress and participates in ER-mediated apoptosis, CHOP may be a key molecule in retinal cell death [17]. In the present study, the phosphorylation of eIF2α was increased concomitantly with the increases in the expression of BiP and CHOP proteins, even through p-eIF2α might be expected to suppress protein synthesis. Boyce et al. [18] reported that selective inhibition of eIF2α dephosphorylation increases both p-eIF2α and CHOP protein. These data suggest that during ER stress, p-eIF2α (inactive form) is still able to stimulate the translation of ATF4 mRNA, thereby increasing the transcription of BiP or CHOP mRNA, but that enough unphosphorylated-eIF2α (active form) may remain to translate BiP and CHOP mRNAs to proteins. On the other hand, we found that staurosporine, which mediates mitochondrial dysfunctions resulting in apoptotic cell death, did not induce any increases in BiP and CHOP proteins in RGC-5 [unpublished data]. Taken together, these findings suggest that persistent ER stress may induce apoptotic cell death through the eIF2α-CHOP signal pathway in RGC-5.
Next, we tried to determine whether tunicamycin could induce retinal damage in vivo. Intravitreal injection of lowdose tunicamycin induced a significant loss of cells in the retinal ganglion cell layer (GCL), but no thinning of the inner plexiform layer (IPL). These findings suggest that retinal ganglion cells are more sensitive to ER stress-induced cell death than other retinal cells. High-dose tunicamycin significantly decreased both the cell count in GCL and the thickness of IPL. The concentration of tunicamycin in the vitreous body after an intravitreal injection of low-dose tunicamycin was estimated to be 10 µg/ml. The tunicamycin concentration achieved within the retina will have been less than this. Interestingly, in the present in vitro study, tunicamycin at 1 to 4 µg/ml induced cell death with an increase in ER-stress signals, suggesting that the in vivo concentration of tunicamycin in the retina was roughly similar to that employed in vitro. Use of tunicamycin at a high dose also led to decreases in IPL, INL (inner nuclear layer), and ONL (outer nuclear layer) in the retina. In guinea pigs, a single subcutaneous injection of tunicamycin at 0.4 mg/kg has been reported to induce hepatotoxicity with dilation of the cisternae of the ER [19]. Fur- thermore, Zinszner et al. [20] noted that in mice, a single sublethal intraperitoneal injection of tunicamycin (1 mg/kg) induces CHOP expression and subsequent severe histological damage with an increase in TUNEL-positive cells, and a characteristic transient renal insufficiency. They also found that CHOP-deficient mice show an attenuated increase in TdTmediated dUTP nick-end labeling (TUNEL)-positive cells during the renal damage induced by tunicamycin. These findings suggest that in vivo, tunicamycin-induced retinal cell death is due, at least in part, to an ER-stress mechanism. NMDA receptors may participate in the processes of excitotoxicity and neuronal death in the retina [21,22]. Previous studies have found that TUNEL-positive cells can be observed in the GCL and INL of the mouse retina at an early stage (within 24 h) after an intravitreal injection of NMDA [23,24]. The hallmark of NMDA-induced neuronal death is a sustained increase in the intracellular Ca 2+ concentration accompanied by overactivation of vital Ca 2+ -dependent cellular enzymes [25]. Thus, the signal-transduction pathways for NMDA-mediated cell death in the retina are well studied, but not yet fully understood.
To illuminate the role and distribution of ER stress in vivo, we focused on the retina of ERAI mice. Information about the status of ER stress during the course of a given disease might be obtained by crossing an ERAI transgenic mouse (the indicator mouse for ER stress in living cells) with a mouse model of the human disease of interest. In flatmounted retinas, fluorescence was detected following various stimulations [tunicamycin, NMDA, and intraocular pressure (IOP) elevation]. To our knowledge, this is the first report demonstrating that NMDA and ischemic insult (elevating IOP), in addition to tunicamycin, can activate the ER stress signal (measured as the splicing of the XBP-1 and venus fusion gene in ERAI transgenic mice) in the retina in vivo. Interestingly, ER stress was also induced in the retina after a transient IOP elevation, defined as an ischemia-reperfusion model. It has been reported that this model exhibits retinal cell damage similar to that induced by NMDA, and that both of these examples of damage are protected against by MK-801, an NMDA receptor antagonist, and by NO synthetase-inhibitor treatment [8,26]. Although little is known about the precise mechanisms responsible for activation of ER stress after NMDA or IOP elevation (ischemia-reperfusion), both stimuli cause intracellular Ca 2+ overload and increased NO production, resulting in apoptotic cell death. Several lines of study suggest that intracellular Ca 2+ overload and excessive production of NO deplete Ca 2+ in the ER, thereby resulting in ER stress [27,28]. Recently, Uehara et al. [10] reported that NO induces S-nitrosylation of proteindisulphide isomerase (PDI), an enzyme that assists in the maturation and transport of unfolded secretory proteins and thereby helps to prevent the neurotoxicity associated with ER stress. S-nitrosylated-PDI exhibits reduced enzymatic activity and induces cell death through the ER stress pathway. These mechanisms may contribute to the activation of ER stress in the retina after NMDA stimulation or IOP elevation. Accordingly, our findings may provide important new insights into the mechanisms underlying the retinal cell damage induced by NMDA and by ischemia-reperfusion. In transverse retinal sections, we observed an increase in fluorescence intensity within the cells of the ganglion cell layer (GCL) and inner plexiform layer (IPL) at 12 and 24 h, respectively, after NMDA injection. The cells displaying increased fluorescence were ganglion cells (at 12 h after the injection), amacrine cells in IPL (at 24 h), and microglia in GCL (at 72 h). These data indicate that ganglion cells may be more sensitive to ER stress than the other retinal cells examined.
To further clarify the participation of ER stress, we examined the changes in BiP and CHOP in the retina after NMDA-induced injury. We found (a) that NMDA induced BiP proteins in the retina at 12 h after its injection (on the basis of immunoblots), and (b) that, NMDA induced both BiP and CHOP in the retina (especially within retinal ganglion cells and INL) at 12 h after its injection (on the basis of our immunostaining results). The expression of the CHOP gene reportedly increases in the rat retina after intravitreal injection of NMDA [29]. Furthermore, Awai et al. [30] found that treatment with MK-801, an NMDA receptor antagonist, inhibited the increases in CHOP mRNA and protein in the mouse retina that are observed after intravitreal injection of NMDA, and moreover that CHOP-deficient mice were resistant to NMDA-induced retinal damage. However, CHOP-deficient mice partially suppressed the NMDA-induced cell death, and therefore other pathways, such as mitochondrial dysfunction, may be engaged in the retinal cell death. Collectively, the above results indicate that NMDA can cause ER stress in the retina, and that the neurotoxicity induced by NMDA is due in part to a mechanism dependent on CHOP protein induction through excessive ER stress.
In conclusion, we have identified a close association between ER stress and retinal damage, and our results suggest that the ER stress-signal pathway might be a good target in the treatment of retinal diseases.